In recent years, home care and community health care in doctor's offices and clinics have improved and the number of early diagnoses and the number of urgent laboratory tests have increased. Against this backdrop, analyzing devices have been demanded that allow quick and easy measurement with high accuracy even if users are not medical technologists. Thus, small analyzing devices for POCT (Point of Care Testing) that can perform precision measurements in a short time without complicated operations have received attention.
Generally, POCT is a generic name for inspections conducted in positions “close to patients”, for example, in consulting rooms of practitioners and specialists, hospitals, and clinics for outpatients. POCT has been a notable method that is quite useful for improving the quality of diagnoses such that a doctor quickly judges an inspection result, immediately performs treatment, and monitors the process of the treatment and the prognosis. Inspections conducted by such small analyzing devices can reduce the cost of transporting specimens, the cost of equipment, and the cost of unnecessary inspections, thereby reducing the total inspection cost as compared with inspections conducted in central inspecting rooms. In the U.S. where rational hospital management is advancing, the POCT market has rapidly expanded and is expected to grow worldwide, including Japan.
In a dry-type biosensor as analysis element typified by an immuno-chromatographic sensor, an adjustment of a reagent is not necessary and an analyte contained in a liquid specimen such as blood and urine to be measured can be analyzed by a simple operation such as dropping the liquid specimen on the biosensor. Currently, a large number of dry-type biosensors have been put into practical use as representative POCT because dry-type biosensors are quite useful for easily and quickly analyzing an analyte in a liquid specimen.
In an immuno-chromatographic sensor (hereinafter abbreviated as a chromatographic sensor) using an antigen-antibody reaction, a sensing reaction is performed with high specificity and a strong binding force. Thus, the chromatographic sensor provides more excellent characteristics than other sensors in an analysis of a physiologically active substance with an extremely low concentration. Currently, using this principle, many diagnostic agents such as pregnancy diagnostic agents, cancer marker diagnostic agents, or cardiac muscle marker diagnostic agents have been commercially available.
As a configuration of a chromatographic sensor, Patent Document 1 discloses an assay device that causes a substance to flow in a plurality of sections (regions) that can communicate with each other by a capillary flow on same plane, that is, on one flat solid support, and determines whether an analyte is present or not in a liquid specimen.
Patent Documents 2 and 3 disclose an analyzing device that detects or measures one of a pair of combined components having immunological binding properties in a liquid.
The above-described methods in Patent Documents 1 to 3 are all described for a configuration of an immunological assay device having a specific reaction, and the device is made of a material such that a liquid specimen can be developed by a capillary force (force caused by capillarity). For example, Patent Document 1 features a configuration such that the flat solid support has a region (first section) in which a tracer that specifically reacts with an analyte is retained, and a region (second section) in which a binder specifically bound to the analyte or the tracer, and the regions (the first section and the second section) are located on the solid support on the same plane and can communicate with each other by a capillary flow. Patent Documents 2 and 3 features a configuration including a sheet-like zone formed of one or more thin pieces placed on the front and rear in a development direction of a liquid specimen and in a contact state where the liquid specimen can be absorbed by each other through edges of the pieces, and a solid support.
An example of a general configuration of a chromatographic sensor typified by Patent Documents 1 to 3 are shown in FIGS. 14A to 14C, 15A, and 15B. A general configuration of the chromatographic sensor includes, on a substrate 1 of a PET sheet or the like, a development flow channel 2, a labeling-reagent retaining portion 4, a specimen application region 5, and a water absorbing portion 6. The development flow channel 2 is formed of a porous carrier such as cellulose nitrate or glass fiber filter paper and partly has a reagent-immobilized portion 3 in which a reagent that specifically reacts with an analyte or a tracer is immobilized. In the labeling-reagent retaining portion 4, a labeling-reagent obtained by labeling a reagent that specifically reacts with an analyte and an immobilized-reagent is retained by a liquid permeable material so that the labeling-reagent is easily dissolved by permeation of the liquid specimen. The specimen application region 5 is provided on the labeling-reagent retaining portion 4 or upstream in a chromatography development direction of the labeling-reagent retaining portion 4, and a liquid specimen is applied on there. The water absorbing portion 6 absorbs water from a developed liquid specimen in a downstream region in the chromatography development direction. On the substrate 1, in order from an upstream side in the chromatography development direction, an upstream end of the labeling-reagent retaining portion 4 is in contact with a downstream end of the specimen application region 5, an upstream end of the development flow channel 2 is in contact with a downstream end of the labeling-reagent retaining portion 4, and an upstream end of the water absorbing portion 6 is in contact with a downstream end of the development flow channel 2. Sensor components other than the substrate 1 (the specimen application region 5, the labeling-reagent retaining portion 4, the development flow channel 2, and the water absorbing portion 6) are all made of a liquid permeable material. In many cases, as shown in FIGS. 15A and 15B, the chromatographic sensor is housed in a hollow casing 51 made of a liquid impermeable material and including a specimen application portion 50 and a result check window 52 so that the reagent-immobilized portion 3 and the specimen application region 5 are partly exposed to the outside.
The development (flow) of the liquid specimen or the like and the measurement principle of the chromatographic sensor will be described. First, the liquid specimen is applied to the specimen application portion 50 in the hollow casing 51. The applied liquid specimen is developed through the specimen application portion 50 while water in the liquid specimen is absorbed by capillarity caused by each construction material for the chromatographic sensor. The liquid specimen developed from the specimen application region 5 and having reached the labeling-reagent retaining portion 4 is further developed downstream while dissolving the labeling-reagent and passes through the reagent-immobilized portion 3 when being developed in the development flow channel 2. The liquid specimen is further developed downstream and water in the liquid specimen is absorbed by the water absorbing portion 6. In case that the liquid specimen contains the analyte, when the analyte reaches the labeling-reagent retaining portion 4, firstly, the analyte undergoes a specific binding reaction with the labeling-reagent to form a “labeling-reagent-analyte complex”. The “labeling-reagent-analyte complex” is developed to the development flow channel 2 together with the liquid specimen, and further reaches the reagent-immobilized portion 3. In the reagent-immobilized portion 3, the “labeling-reagent-analyte complex” also undergoes a specific binding reaction with the immobilized-reagent to form a “labeling-reagent-analyte-immobilized-reagent complex”. Then, in the reagent-immobilized portion 3, a color reaction caused by the labeling-reagent depending on the concentration of the analyte can be checked through the result check window 52. The general chromatographic sensor typified by this configuration is widely distributed for use in hospitals or home in the market. Herein, a sandwich reaction that forms the “labeling-reagent-analyte-immobilized-reagent complex” is taken as an example of an immune reaction for describing the measurement principle, but a competitive reaction is often used.
In the configuration of the chromatographic sensor, in order to help development of the liquid specimen and retaining the developed liquid specimen, the water absorbing portion 6 made of a material that satisfactorily absorbs liquids is provided adjacent to the development flow channel 2. Also, it is important that when the liquid specimen is applied, the labeling-reagent is discharged from the labeling-reagent retaining portion 4 as quickly and completely as possible, and developed with the development of the liquid. Thus, it has been proved that a porous material or a fiber material is suitable as a construction material for the labeling-reagent retaining portion 4. As a specific example, the labeling-reagent retaining portion 4 is often made of paper, fleece, a porous plastic layer, or a membrane. However, since the labeling-reagent retained by the construction material is permeated to a deep part of the material, a sufficient amount of liquid specimen is often difficult to dissolve. Also, since the construction material and the labeling-reagent are nonspecifically adsorbed during a dry state, the retained labeling-reagent cannot be completely dissolved, and the degree of dissolution of the labeling-reagent differs depending on chromatographic sensors. Thus, when quantitative measurement using the immuno-chromatographic sensor is performed, unfortunately, sufficient accuracy cannot be obtained and measurement with high sensitivity cannot be performed.
Next, it is described with reference to FIGS. 16A to 16F (in FIGS. 16B to 16F, only the chromatographic sensor is shown and the hollow casing 51 is omitted) that a state where the liquid specimen is developed on a conventional general chromatographic sensor shown in FIGS. 15A and 15B when the liquid specimen is applied to the chromatographic sensor. First, as shown in FIGS. 15A and 16A, a liquid specimen (in FIGS. 15A and 16A, a blood sample S) is applied to the specimen application portion 50 using a tool such as a dispenser or a dropper (or a syringe 54) (Step 1). Then, as shown in FIG. 16B, the liquid specimen permeates and spreads over the entire specimen application region 5 (Step 2). Then, as shown in FIG. 16C, the liquid specimen retained in the specimen application region 5 is developed to the labeling-reagent retaining portion 4 (Step 3). At this time, a labeling-reagent coming into contact with a development tip of the liquid specimen is quickly dissolved, and as shown in FIG. 16D, the dissolved labeling-reagent is developed with the flow of the liquid specimen. During the development of the liquid specimen, an analyte and the labeling-reagent form a complex, and the complex is developed with the flow. The labeling-reagent is swept by the liquid specimen and dissolved. As shown in FIG. 16E, the labeling-reagent is developed in the development flow channel 2 and passes through the reagent-immobilized portion 3. In Step 5, as shown in FIG. 16F, the labeling-reagent is developed to the water absorbing portion 6 and absorbed (Step 6). As such, the labeling-reagent exists with higher concentration in positions closer to the development tip of the liquid specimen. The analyte that can come into contact with the labeling-reagent to form a complex mostly exists in a development front portion in the liquid specimen, while there is little labeling-reagent in the liquid specimen in a development rear portion and only an unreacted analyte is developed.
Thus, for the above-described configuration of the chromatographic sensor, the entire liquid specimen is actually not evaluated by the chromatographic sensor as a result, but the existence or the amount of the analyte in a part of the applied liquid specimen is simply determined. Thus, when the analyte with a high concentration exists in the liquid specimen, the analyte can be sufficiently detected, but when the analyte with a low concentration exists, the analyte can be detected if all the labeling-reagents and the analyte extremely satisfactorily react with each other. However, for the above reason, it is impossible to satisfactorily detect the analyte with the chromatographic sensor having the current configuration, and unfortunately, a region with high sensitivity (a nanomole region or a picomole region to a lower concentration region) of an analyte cannot be accommodated.
As described above, in the conventional configuration, the labeling-reagent cannot be completely dissolved and is not uniformly diffused and developed in the liquid specimen. Thus, when quantitative measurement using the chromatographic sensor is performed, unfortunately, it is impossible to obtain sufficient accuracy and perform measurement with high sensitivity.
Further, since all the sensor components other than the substrate 1 are formed of a liquid permeable material such as glass fiber filter paper or a porous film, the liquid specimen permeates each sensor component and water in the liquid specimen is retained, thereby causing a liquid amount (dead volume) that does not flow out downstream. Thus, in the conventional chromatographic sensor, a larger amount of liquid specimen needs to be applied in view of at least the dead volume due to the water retained by each sensor component. Thus, when the liquid specimen is, for example, blood, it is impossible to measure the blood as a small amount of specimen.
Patent Document 4 reports a biosensor including a space forming portion in which a clearance as a space for the flow by capillarity is formed in a part of a development layer. Herein, the space forming portion is formed such that a space in a specimen application portion is enclosed by a liquid impermeable material, and can retain a certain amount of liquid specimen. However, a labeling-reagent is formed in a part of the development layer and thus the amount of specimen cannot be sufficiently reduced. Also, a dissolution pattern of the labeling-reagent by the development of the liquid specimen is the same as in Patent Documents 1 to 3. Thus, the dissolution and development states of the labeling-reagent in Patent Documents 1 to 3 cannot be improved.